Project summary/abstract Stith, Bradley James Recently, we reported that agonist elevation of the lipid phosphatidic acid (PA) leads to Src tyrosine kinase activation, and then Src phosphorylation and activation of substrate phospholipase C-gamma (PLC?). Using an in vivo Xenopus animal model system for Src regulation and membrane translocation, we have developed a two-stage mechanism of activation of Src: (1) anchoring of inactive Src to membrane rafts by the nonspecific binding of 1 or more anionic lipids to the SH4 domain of Src. (2) After PA elevates, the lipid binds to two other domains called the SH3 and ULBR domains. The binding of PA would change the conformation of Src to ?unravel? the ?bound up kinase? and this would increase the tyrosine kinase activity. This model is based on our knowledge of how Src is unraveled by regulatory proteins, and our lipid binding studies along with those of Drs. J. Falke (University of Colorado Boulder), S. McLaughlin (SUNY Stony Brook) and collaborator Dr. M. Pons (University of Barcelona) on both Src and protein kinase C. Our ?broad, long-term objectives? involve lipid and tyrosine kinase regulation of cellular processes and their roles in disease. More specifically, PA and Src have been associated with cell proliferation, migration, autophagy, neurodegenerative diseases, and platelet aggregation. Thus, our work may impact diseases such as cancer (both PA and Src are elevated in tumor cells), metastasis, autophagy associated diseases, Alzheimer's disease and stroke. With the help of two collaborators, we will use a new technique (TIRF microscopy) that measures lipid- human Src binding to examine the accuracy of our 2 step model. As described in the proposal, this new method for studying lipid regulation of proteins offers many advantages over older methods. Recently, Dr. Falke has used TIRF microscopy to help define the mechanism of lipid activation of protein kinase C. We may also find proof that PA forms large membrane patches that may cluster Src (and provide another method of Src regulation). These studies will examine whether elevated [Ca]i or Src phosphorylation can displace Src from membranes. This displacement would be a mechanism to inactivate Src or alter the substrate availability of the cytoplasmic Src (i.e., changing Src's actions). We will also use phosphospecific western blotting to determine the in vivo changes in regulatory phosphorylation of Src after agonist or lipid (e.g., PA and certain other anionic lipids that we have shown bind to Src) addition. To facilitate these studies, we will draw upon Dr. Stith's 25 years of lipid signaling and tyrosine kinase regulation, and my 2 collaborators, to define lipid regulation of Src tyrosine kinase.